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  System for staining fungi and protozoaRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Viable Micro-organism, Determining Presence Or Kind Of Micro-organism; Use Of Selective MediaThe Patent Description & Claims data below is from USPTO Patent Application 20070031917. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a Continuation-in-Part of, and claims priority to currently pending U.S. patent application Ser. No. 10/249,663, entitled, "Method for Staining Fungi and Protozoa", filed Apr. 29, 2003, the contents of which are incorporated herein by reference, which claims the benefit of U.S. Provisional Patent Application 60/319,214, entitled, "Method for Staining Fungi and Protozoa", filed Apr. 29, 2002. BACKGROUND OF THE INVENTION [0002] This invention is a novel method of staining and detecting protozoa and fungi for observation under a light microscope, and with application to the qualitative and quantitative detection of microsporidia in environmental water samples. [0003] Fungi and microsporidia can be differentiated from other microorganisms based on their chitin content. Current dyes used to stain chitin in fungi and microsporidia are based on fluorescent optical brighteners (Review by Ruchel et al., 2001), requiring a fluorescent microscope for observation. Details are not observable by this method. [0004] A need exists in the art for the identification of the characteristic structures in microsporidian spores, including but not limited to thick spore walls, vacuoles, beltlike stripes and sporoplasms, that can be implemented using a light microscope, and recorded using a digital camera. SUMMARY OF INVENTION [0005] The present invention provides a method for determining the presence or absence of a microorganism in a sample suspected of containing a microorganism. In a preferred embodiment, a sample is provided to be analyzed, the sample is treated with a protein stain, and the sample is then treated with a cationic carbocyanine dye and examined for microorganisms. [0006] In an additional embodiment, the protein stain used to treat the sample is Ponceau S, and preferably is a solution of 0.1% Ponceau S in 5% acetic acid in water. [0007] In an additional embodiment, the cationic carbocyanine dye used to treat the sample is Stains-all, and preferably is a solution of Stains-all in methanol diluted 1:10 in a solution of deionized water, acetic acid, and methanol at 50:10:40. [0008] In an additional embodiment of the present invention, an additional step of selectively decolorizing the sample is included. Selectively decolorizing the sample is facilitated through the application of a decolorizing solution. The decolorizing solution can be a solution of sodium dodecyl sulfate in phosphate buffered saline. Preferably the sodium dodecyl sulfate being 0.25% in phosphate buffered saline. Additionally, the decolorizing solution can be a solution of acid methanol, in a preferred solution of 50:10:40 deionized water, acetic acid, and methanol. [0009] In an additional preferred embodiment of the present invention, an additional step of rinsing the sample prior to examining the sample in included. The rinsing step is facilitated by the application of a solution of acid methanol to the sample and subsequent treatment of the sample with a solution of phosphate buffered saline. Preferably the solution of acid methanol employed in the rinsing step is 50:10:40 deionized water, acetic acid, and methanol and the solution of phosphate buffered saline contains about 0.05% Tween 20. [0010] In an additional embodiment, the decolorizing solution is 6M guanidine hydrochloride. In yet another embodiment, the decolorizing solution is about 0.3% Tween 20. [0011] In a preferred method of the present invention for the determination of the absence or presence of a microorganism in a sample suspected of containing a microorganism, a provided sample is treated with a solution of Ponceau S and Stains-all dye solution. The sample is then selectively decolorized, rinsed and examined for microorganisms. [0012] In another embodiment, the step of selectively decolorizing the sample further includes the steps of applying three decolorizing solutions to the sample and rinsing the sample after each application. [0013] A kit for detecting the presence of a microorganism is provided by the present invention in which is provided a protein stain and a cationic carbocyanine dyne. [0014] In an additional embodiment of the kit, the protein stain is Ponceau S, and preferably a solution of 0.1% Ponceau S in 5% acetic acid in water. [0015] In yet another embodiment of the kit of the present invention, the cationic carbocyanine dye is Stains-all, and preferably a solution of Stains-all in methanol diluted 1:10 in a solution of deionized water, acetic acid, and methanol at 50:10:40. [0016] In an additional embodiment, the kit of the present invention includes a selective decolorizing solution. The decolorizing solution being sodium dodecyl sulfate in phosphate buffered saline, and preferably a solution of 0.25% sodium dodecyl sulfate in phosphate buffered saline. Additionally, the decolorizing solution can be a solution of acid methanol, preferably a solution of 50:10:40 deionized water, acetic acid, and methanol. [0017] In yet another embodiment, the kit of the present invention includes a rinsing solution. The rinsing solution being acid methanol, preferably the solution of acid methanol is 50:10:40 deionized water, acetic acid, and methanol. [0018] In another embodiment, the kit contains phosphate buffered saline solution. Preferably, the phosphate buffered saline solution being 0.05% Tween 20. BRIEF DESCRIPTION OF THE DRAWINGS [0019] For a fuller understanding of the nature and objects of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which: [0020] FIG. 1 are photographic slides of the staining of Penicillium notatum using the present invention. Continue reading... Full patent description for System for staining fungi and protozoa Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this System for staining fungi and protozoa patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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